Page 33 - Power of Stem Cells- arthritis and regeneration

www.nature.com/scientificreports/

Figure 3. MSC reduces CIA-associated increases in NF-κB activities in synovial fibroblasts. (A,B) The
nuclear NF-κ B (p65) levels (A) and cytoplasmic Iκ B levels (B) in synovial fibroblasts in CIA-mice treated with
MSCs (MSCs), or without MSCs (saline). (C) The mRNA levels of Iκ B in synovial fibroblasts. * p < 0.05. NS:
non-significant. N = 5.

anti-NF-κ B (p65; detection of total protein; Cell Signaling, San Jose, CA, USA; Catalog number: 8242 s; diluted
1:1000), rabbit anti-Iκ B (Cell Signaling; Catalog number: 4812 s; diluted 1:1000), anti-LaminB1 (Cell Signaling;
Catalog number: 12586 s; diluted 1:1000) and anti-α -tubulin (Cell Signaling; Catalog number: 2125 s; diluted
1:1000). Secondary antibody is HRP-conjugated anti-rabbit (Dako, Carpinteria, CA, USA; Catalog number:
P0448; diluted 1:1000). Figure images were representative from 5 repeats. LaminB1 was used as a protein loading
control for nuclear protein, and α -tubulin was used a protein loading control for cytoplasmic protein.

Isolation and culture of synovial fibroblasts. For isolation of human synovial fibroblasts, the healthy
joint tissue from a deceased 55-year-old male was dissected and placed in separate large petri dishes. For isolation
of mouse synovial fibroblasts, the joint tissue was obtained from male DBA/1J mice at 8 weeks of age. A scalpel
was used to cut tissue pieces as small as possible. Thereafter, the tissue pieces were carried over to a new 10 ml
tube, and 2.5 ml 30 mg/ml collagenase (Sigma-Aldrich) was added to the tissue to incubate for 60 min at 37 °C.
In the last 10 minutes of this 60 minute incubation, 5 ml 0.25% trypsin (Sigma-Aldrich) was added to the tube.
Afterwards, the tube was centrifuged for 5 minutes at 300 g to pellet the cells. The cells were then re-suspended
and seeded into a six-well plate that was pre-coated with a 0.1% gelatin solution for 20 minutes at 37 °C. The gela-
tin was removed from the dish before the cells were seeded. Passaging of the cells was performed when confluence
of the culture plate was achieved. The culture media for human or mouse synovial fibroblasts consisted of DMEM
(Gibco) supplemented with 15% FBS. Transfection of the synovial fibroblasts was performed by Lipofectamine
2000 reagent (Invitrogen, Carlsbad, CA, USA) for 24 hours.
Luciferase-reporter activity assay. Luciferase-reporters were successfully constructed using molec-
ular cloning technology. The target sequence was inserted into a pGL3-Basic vector (Promega, Madison, WI,
USA) to obtain pGL3-Iκ B-3′ UTR, which contained the miR-548e binding sequence (Iκ B-3′ UTR sequence).
miR-548e-modified fibroblasts were seeded in 24-well plates for 24 hours, after which they were transfected with

Scientific RepoRts | 6:28915 | DOI: 10.1038/srep28915 5